Specimen Preparation for TEM

Specimens for TEM are often deposited on or in a thin (10 to 20 nm) membrane that rests on a grid. These grids are usually made of copper and 

Since most materials are opaque to the electron beam, even when only a few hundred nanometers thick, special problems arise in the production of suitable mounted specimens. Specimen support films are usually made of plastic or carbon, though other materials have also been used. Suitable film solutions may be made up of 2% w/v formvar (polyvinyl formal) in ethylene dichloride or chloroform. 

An alternative procedure is to clean a microscope slide with detergent and polish with a clean cloth without rinsing away the detergent, so as to form a hydrophilic layer at the surface, which facilitates stripping. The slide is dipped in a solution of formvar in ethylene dichloride (0.3% to 0.7% depending on the film thickness required) and allowed to drain dry. The film may be floated on to a water surface and mounted on grids as before. If individual grids are required, the film may be cut into small 

Another method of preparing the sample is to disperse it in linseed oil, which is then thinned with white spirit. The dispersion is spread over a microscope slide that is immersed in white spirit for a few minutes to remove the oil. After drying the slide a thin layer of carbon is deposited on the specimen to form a supporting film. Finally this is floated off on water as before and picked up on a grid for examination [91 ]. 

A dispersed sample may also be obtained by means of an aerosol sampling device. A suitable technique is to form a sandwich of plastic film particles and 20 nm thick carbon. The underlying plastic may then be washed away with solvent and the specimen examined after shadowing 159,160,162]. 

The most comprehensive textbook on TEM is that by Williams and Carter (1996). It is written for the microscope operator, and, as well as explaining the use of the instrument for imaging, diffraction and spectroscopy, it also gives an account of the principles of specimen production, and the reader is encouraged to turn to that book for a fuller account of the subject. 

The final TEM specimen must of course be electron transparent, and it must be either self-supporting or it must rest upon some form of support grid (usually made of copper). The specimen or grid will usually be 3 mm in diameter. 

Self-supporting disks are prepared in several stages an initial slice of material is prepared between 100 pm and 200 pm in thickness. Ductile materials may be rolled, cut with a chemical saw, or spark eroded to produce a thin slice brittle materials may be cleaved with a razor blade or cut with an ultramicrotome (see below). The 3 mm disk is then cut from the slice, using a mechanical punch for ductile materials, while brittle materials need to be spark eroded or drilled. 

Specimens on grids are an alternative to self-supporting disks. Specimens in the form of small particles may be supported on a thin film (e.g. an amorphous carbon film) before being placed on the grid. Thin slices of the material may be produced by an instrument known as an ultramicrotome. The principal advantage of the technique is that it leaves the chemistry unchanged, and it may be employed to create uniform thin films of multiphase material. The instrument operates by moving 

In a lOOkV TEM, useful specimens can range in thickness from 20nm to 0.5 pm. For micro analysis, the thinner the specimen the better the quality of the information that can generally be obtained. 

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Fig. 2 Schematic of embedding and sectioning procedure used to limit relaxation of crack-tip microdeformation during specimen preparation for TEM (here for a CT specimen deformed under static loading)...

D. Bahnck and R. Hull, Experimental measurement of transmission electron microscope specimen temperature during ion milling, in R. Anderson, ed., Specimen Preparation for TEM, Vol. II, MRS Vol. 199, Materials Research Society, Warrendale, PA, 1990. 

The surface areas of dust samples as determined by optical and electron microscope have also been compared [167]. Pore size distributions of thin films of AI2O3, as measured by TEM, have also been compared with those determined by gas adsorption/desorption [168]. It has also been suggested that electron microscope gives a truer estimate of surface area than gas adsorption techniques [169]. Further information can be obtained in a recent review of specimen preparation for TEM [170]. 

Disadvantages of TEM depend on the researcher s point of view. Since the TEM operates under high vacuum conditions, live specimens cannot be imaged. In addition, specimen preparation for TEM is not trivial. In fact, in some cases, it is as much art as technology. In general, proper specimen preparation requires both auxiliary equipment and expertise. The more complex the research questions, the more complex and tedious the specimen preparation. This, however, can be approached - from a more positive stance. The vast capabilities of the TEM can be exploited by the selection of specific specimen preparation protocols that are suited to specific experimental designs. 

Interfacing Light Microscopy with TEM Fixation and Specimen Preparation for TEM Selective Staining of Nuclear Components Image Analysis and Enhancement Three-Dimensional Reconstructions by TEM... 

Specimen preparation for TEM generally in-voles the formation of a thin film of the material less than 100 nm thick. The methods used for this preparation depend upon the nature of the polymer and its physical form. In the case of thick or bulk specimens, microtomy is generally used. In the case of solutions, powders or particulates, simpler methods can provide a thin, dispersed form of the material. Three types of simple preparations will be described later in this section dispersion, disintegration and film casting. The more complex methods such as microtomy, replication, etching and staining will be described in other sections of this chapter. 

Bassett DC, Olley RH, Vaughan AS (2003) Specimen preparation for TEM of polymers. In Pethrick RA, Viney C (eds) Techniques in polymer organisation and morphology characterisation. Experimental methods in polymer characterisation. Wiley, Chichester Bovey FA (2007) NMR of polymers. eMagRes. Wiley Online... 

Transmission electron microscopy is very widely used by biologists as well as materials scientists. The advantage of being able to resolve 0.2 nm outweighs the disadvantages of TEM. The disadvantages include the inabiUty of the common 100-kV electron beam to penetrate more than a few tenths of a micrometer (a 1000-kV beam, rarely used, penetrates specimens about 10 times thicker). Specimen preparation for the TEM is difficult because of the... 

Figure 11.8. Illustration of sample preparation for cryo-TEM imaging. Parts (a)-(d) show specimen preparation for sample freezing (e) shows plunge freezing apparatus with temperature and humidity control and ( ) a typical image obtained by cryo-TEM. Copyright 2006 Nestec Etd.

Imaging with the AFM does not require that the specimen be conductive. For this reason, the AFM offers greater potential for biological samples them does STM, although there are still challenges inherent in the design of the tips and the cantilevers on which they are borne (140). Low-temperature methods, under development, may prove to be as reliable a method of specimen preparation for the AFM as it has been for the TEM. 

Each view provides a different perspective on the membrane structure while, together, they give the complete structural model. Specimen preparation for OM and TEM cross sections was by nucrotomy of embedded membrane strips using a method developed to limit structural collapse (Section 4.3.4). An optical micrograph (Fig. 5.28A) shows the membrane cast on a woven support fabric. The active surface layer (top)... 

Replication is one of the oldest methods used for the production of thin TEM specimens and it is also used for specimen preparation for optical microscopy and SEM. The procedure was first introduced by Bradley [426, 427], and it is well documented in texts on specimen preparation [428, 429]. Replicas have the surface characteristics or topography of the original specimen without the need to directly image beam sensitive materials. Reflected light microscopy of polymer surfaces is often difficult because glare from the surface limits visible detail, although... 

Recently, Shibayama who is co-author of this paper has successfully developed a nano-mechanics in-situ Transmission Electron Miaoscope (TEM) experimental apparatus as shown in Figure 1, and the applied load - indentation depth (displacement) curve can be directly measured during the in-situ observation. In order to study the fracture behavior at the interface between SiC fiber and matrix, a miniaturized Double Notched Shear (DNS) specimen of NITE (Nano-powder Infiltration and Transient Eutectic Process) SiC/SiC composite was prepared for TEM by Focused Iron Beam (FIB) equipment. The inter-laminar shear strength was estimated as 2.8 x 10 MPa, which is about thirty times higher than the result obtained by the conventional DNS tesL In addition, our numerical analysis using the interface element " ... 

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